CN113533552B - Method for synchronously detecting ARR (acute respiratory syndrome) drugs in process of detecting renin activity by liquid chromatography-tandem mass spectrometry - Google Patents

Method for synchronously detecting ARR (acute respiratory syndrome) drugs in process of detecting renin activity by liquid chromatography-tandem mass spectrometry Download PDF

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CN113533552B
CN113533552B CN202110637441.2A CN202110637441A CN113533552B CN 113533552 B CN113533552 B CN 113533552B CN 202110637441 A CN202110637441 A CN 202110637441A CN 113533552 B CN113533552 B CN 113533552B
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刘鹏云
孔子青
马金飞
袁小芬
刘华芬
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Kailaipu Technology Co ltd
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Hangzhou Calibra Diagnostics Co ltd
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Abstract

The application provides a method for synchronously detecting therapeutic drugs affecting ARR (ratio of plasma aldosterone to renin activity) in the process of detecting renin activity by liquid chromatography tandem mass spectrometry, which realizes qualitative screening of drugs affecting ARR values while detecting plasma renin activity by liquid chromatography tandem mass spectrometry, and can be used for assisting in analysis and judgment of whether ARR is negative, positive, false negative or false positive. The effective extraction of angiotensin I and 43 antihypertensive drugs is realized while the protein precipitation is carried out on the sample; the characteristics of high flux, high specificity and high sensitivity of the liquid chromatography tandem mass spectrometry technology are utilized to synchronously detect multiple indexes including angiotensin I, 43 antihypertensive drugs and the like on an extracted sample; and (3) performing primary screening of the medicine by using MRM mass spectrum parameters of the screened medicine, and rechecking according to the retention time of the medicine. ARR detection values are jointly analyzed in combination with drug screening results, false positive or false negative results of detection are effectively distinguished while stopping drug treatment of patients, accuracy of actual primary aldosteronism and clinical renin activity detection is improved, and clinical popularization and application are facilitated.

Description

Method for synchronously detecting ARR (acute respiratory syndrome) drugs in process of detecting renin activity by liquid chromatography-tandem mass spectrometry
The application claims priority of China prior application, application number 2021106145678, application day 2021, 6 and 2; all of which are included as part of the present application.
Technical Field
The application relates to the technical field of chemical analysis, in particular to a qualitative detection method for primary aldosteronism, and especially relates to a detection and judgment method for the ratio ARR of Plasma Aldosteronism Concentration (PAC)/Plasma Renin Activity (PRA).
Background
Primary aldosteronism (PA, abbreviated as orthoaldehyde), is a common endocrine hypertension characterized by spontaneous secretion of aldosterone by the adrenal cortex, leading to internal retention of sodium and potassium, increased blood volume, and inhibited renin-angiotensin system activity, which is clinically manifested mainly as hypertension and hypokalemia.
The ratio ARR of Plasma Aldosterone Concentration (PAC)/Plasma Renin Activity (PRA) is the most reliable method of screening for primary aldosteronism. Among them, plasma renin activity is an index for measuring the production efficiency of angiotensin I in peripheral blood under renin catalysis, and is of great importance in the genotyping diagnosis of hypertension caused by Renin Angiotensin Aldosterone System (RAAS).
In 2010, the department of endocrinology was drawn by the society of Chinese medicine, and screening of procyanidins was performed on 1656 refractory hypertensive patients in 11 provinces and 19 centers nationwide, and the prevalence was reported to be 7.1%. Li Qifu professor team published studies indicate that the incidence of orthoaldehydes in newly diagnosed hypertension exceeds 4.0%. ARR is used as a first-choice screening index of the protoaldosis in 2020 edition of expert consensus on diagnosis and treatment of primary aldosteronism, and screening of the protoaldosis for hypertension, particularly refractory hypertension and newly diagnosed hypertension groups is considered to have practical guiding significance for clinical work, and the protoaldosis screening for all newly diagnosed hypertension patients is recommended.
It is further pointed out in expert consensus that ARR values are affected by a number of factors including age, sex, diet, medication, posture, potassium and creatinine, and may lead to false positive or false negative results, where particular attention is paid to the therapeutic drug used. Consensus requires that detection of ARR should be performed at least four weeks after the withdrawal of drugs with a greater impact on ARR number to avoid false positive or false negative results; of particular note are ACE inhibitors (ACE-Is), dihydropyridine Calcium Channel Blockers (CCBs) and angiotensin ii receptor blockers (ARBs), which can be detected by lowering PAC and greatly increasing PRA levels to lower ARR, resulting in false negative detection results, requiring at least 2 weeks of discontinuation of the drug. However, as orthoaldehydes often cause severe hypertension, withdrawal is potentially harmful to the patient.
ARR assays, including Plasma Aldosterone (PAC) and Plasma Renin Activity (PRA), currently employ radioimmunoassay to measure PRA, indirectly reflecting the level of renin activity in the plasma by the rate of conversion of angiotensinogen to angiotensin I per unit volume per unit time. Most centers also use radioimmunoassay to determine blood aldosterone (PAC). The immunological method for measuring the activity of renin and the content of aldosterone has the defects of inherent poor specificity, cross coupling reaction and the like of the immunological method, and can not realize the screening of the antihypertensive therapeutic drug influencing the ARR value, and patients need to face the risk of drug withdrawal detection.
Therefore, it is urgently needed to find a simpler and more convenient ARR detection method, which can not only avoid stopping drug treatment for patients, but also effectively reduce false positive or false negative results of primary aldosteronism detection, and improve the accuracy of clinical actual ARR and renin activity detection.
Disclosure of Invention
Aiming at the problems in the prior art, the application provides a synchronous qualitative detection method for the antihypertensive drugs affecting ARR in the process of detecting the activity of renin by a liquid chromatography tandem mass spectrometry method, which mainly realizes qualitative screening of the drugs affecting the ARR value while detecting the activity of plasma renin by the liquid chromatography tandem mass spectrometry. ARR detection values are jointly analyzed by interfering with drug screening results, so that the patient is prevented from stopping drug treatment, meanwhile, false positive or false negative results of detection are effectively reduced, the accuracy of actual primary aldosteronism and clinical renin activity detection is improved, and clinical popularization and application are facilitated.
On the one hand, the application provides a synchronous qualitative detection method for the antihypertensive drugs affecting ARR in the process of detecting the activity of renin by a liquid chromatography-tandem mass spectrometry method, which mainly realizes qualitative screening of the antihypertensive drugs affecting ARR in blood plasma while detecting the content of angiotensin I in a blood plasma sample, combines the positive screening drugs to affect the ARR and combines the concentration of aldosterone, and can be used for comprehensively judging and analyzing whether the ARR is likely to be one of negative, positive, false negative or false positive.
Further, the total 43 antihypertensive drugs affecting ARR include nine beta-blockers, five potassium-excreting diuretics, three potassium-retaining diuretics, ten angiotensin converting enzyme inhibitors, six angiotensin receptor antagonists, eight calcium antagonists, one central alpha 2 receptor agonist and one nonsteroidal anti-inflammatory drug; the ARR has a calculation formula as follows: ARR = aldosterone concentration/angiotensin I production rate.
According to 2020 edition expert consensus on diagnosis and treatment of primary aldosteronism, the reasons for drugs that lead to false positives or false negatives of ARR are shown in the following table:
note that: ARR: the ratio of plasma aldosterone to renin activity; ACEI: angiotensin converting enzyme inhibitors; ARB: angiotensin receptor antagonists; CCB: calcium ion antagonists
And in 2020 edition of expert consensus on diagnosis and treatment of primary aldosteronism, it is prescribed that the measurement of ARR should be performed at least four weeks after the administration of drugs having a large influence on the ARR value, so as to avoid false positive or false negative results, and that the administration of drugs such as ACEI, ARB and CCB should be performed again at least 2 weeks after the administration of drugs to avoid false negative results. However, as orthoaldehydes often cause severe hypertension, withdrawal is potentially harmful to the patient.
According to the application, through market research, 43 hypertension therapeutic drugs which have great influence on ARR and have high use frequency are collected, and a liquid chromatography tandem mass spectrometry analysis method is established, so that the drugs affecting ARR are creatively and synchronously detected in the renin activity detection process, the risk of stopping drugs is avoided, the accuracy of clinical actual ARR judgment is improved, the false positive or false negative result of primary aldosteronism detection is effectively reduced, and the clinical application significance is great.
Plasma Aldosterone Concentration (PAC) in the ARR detection process of the application adopts solid-liquid extraction (SLE) to pretreat a plasma sample, and liquid chromatography tandem mass spectrometry technology is used for detection.
Further, the antihypertensive drugs affecting ARR are respectively:
the nine beta-receptor blockers comprise alolol, atenolol, bisoprolol, esmolol, labetalol, metoprolol, nebivolol, propranolol and sotalol;
the five potassium-expelling diuretics comprise bumetanide, furosemide, hydrochlorothiazide, indapamide and torsemide;
the three potassium-retaining diuretics comprise amiloride, eplerenone and spironolactone;
the ten angiotensin converting enzyme inhibitors comprise benazepril, captopril, enalapril, fosinopril, imidapril, lisinopril, perindopril, quinapril, ramipril, trandolapril;
the six angiotensin receptor antagonists include candesartan, irbesartan, losartan, olmesartan, telmisartan, and valsartan;
the eight calcium ion antagonists comprise amlodipine, benidipine, felodipine, flunarizine, lercanidipine, nicardipine, nifedipine and nimodipine;
the one central α2 receptor agonist comprises methyldopa;
the non-steroidal anti-inflammatory drug includes aspirin.
Further, the beta-blocker, the central alpha 2 receptor agonist and the nonsteroidal anti-inflammatory drug can all raise ARR, and a false positive result appears; the potassium-expelling diuretic, potassium-retaining diuretic, angiotensin converting enzyme inhibitor, angiotensin receptor antagonist and calcium ion antagonist can reduce ARR, and false negative result appears.
Any of the beta-blocker, central alpha 2 receptor agonist, non-steroidal anti-inflammatory drug can decrease the aldosterone concentration and renin activity in the patient, wherein the renin activity decreases to a greater extent than the aldosterone concentration, thereby causing a false positive result.
A potassium-excreting diuretic or a potassium-retaining diuretic is capable of elevating the patient's aldosterone concentration and renin activity, wherein the renin activity is elevated to a greater extent than the aldosterone concentration; any one of the angiotensin converting enzyme inhibitor, the angiotensin receptor antagonist and the calcium ion antagonist can reduce the aldosterone concentration of the patient, and the renin activity can be increased, thereby generating false negative results.
Further, the step of analyzing the value of ARR is: analyzing and judging ARR as one of negative, positive, false negative or false positive through a clinical medication AI guiding system; the clinical medication AI guiding system comprises a patient information module, a database module and a decision tree system.
Further, the information module is used for recording basic information of a patient; the database module is used for storing the detection result of ARR and the detection result of the hypertension treatment drug; the decision tree system automatically analyzes and judges the ARR index as one of negative, positive, false negative or false positive according to the detection result.
Further, when the primary aldosteronism is determined to be one of negative, positive, false negative, or false positive according to the analysis of the detection result, the determination tangent point value of the ARR is 30.
Further, the analysis and judgment method of the decision tree system comprises the following steps: when the detection result of the ARR is a negative result slightly lower than 30, if the patient contains the drug which can reduce the ARR, the patient is likely to be false negative and needs to stop drug recheck or further carry out a diagnosis experiment of the original aldehyde; when the detection result of ARR is a negative result lower than 30, if the patient contains the medicine capable of raising ARR, the patient can be judged as negative; when the detection result of the ARR is a positive result higher than 30, if the patient contains the medicine capable of raising the ARR, the patient is likely to be false positive, and the medicine stopping review or the orthoaldehyde diagnosis experiment is further carried out; when the result of detection of ARR is a positive result of more than 30, it can be judged as positive if the patient contains a drug capable of lowering ARR at the same time.
Further, the angiotensin I production rate is determined by measuring the concentration of angiotensin I in the sample before and after incubation according to the formula: angiotensin i production rate = (angiotensin i concentration after incubation-angiotensin i concentration before incubation)/incubation time;
further, the specific detection steps of the qualitative detection method for the primary aldosteronism are as follows:
(1) Sample pretreatment: 1) Taking two 100 mu L samples to be tested in parallel, respectively adding generating buffer solution into the samples, uniformly mixing, incubating one sample, and directly carrying out the next operation without incubating the other sample; 2) Adding a reaction stopping solution into the sample added with the generated buffer solution and uniformly mixing; 3) Adding an internal standard-containing protein precipitant into the sample added with the reaction stopping solution in the step 2) and uniformly mixing by vortex; 4) Uniformly mixing the sample added with the protein precipitant in the step 3), centrifuging, and taking supernatant for machine detection;
(2) Adopting a high performance liquid chromatography tandem mass spectrometry system to detect the angiotensin I of the solution to be detected obtained in the steps, and synchronously screening 43 hypertensive therapeutic drugs affecting ARR by using an unoccupied sample;
(3) For the samples which are positive according to the therapeutic drug mass spectrum parameter screening result in the step (2), the retention time of the standard substances of all the hypertension therapeutic drugs in the drug mixed working solution is required to be controlled simultaneously, and whether the drug screening result is false positive is checked, so that the accuracy of the drug screening positive result is ensured;
the protein precipitant containing the internal standard is as follows: preparing a protein sedimentation agent containing an internal standard by using methanol, a zinc sulfate aqueous solution and an angiotensin I isotope internal standard;
the generating buffer solution is as follows: PMSF-containing Tris and EDTA buffers;
the reaction termination liquid is as follows: formic acid or acetic acid;
the screening medicine mixed working solution comprises the following components: the mixed working solution of the screening medicaments for detecting the lower limit concentration is prepared by using 43 standard substances of the hypertension therapeutic medicaments affecting the ARR.
Further, the standard curve is prepared: preparing standard curves with different concentrations by using an angiotensin I standard;
at present, a radioimmunoassay is generally adopted for ARR detection, and screening of various therapeutic drugs cannot be achieved through single detection by the radioimmunoassay, so that if a patient does not stop drug according to a rule before ARR detection is carried out, the influence of a antihypertensive drug on ARR detection is difficult to evaluate.
According to the application, a great number of researches prove that 43 hypertension therapeutic drugs affecting ARR can be synchronously detected when the Plasma Renin Activity (PRA) is detected through liquid chromatography-tandem mass spectrometry, more accurate judgment of the ARR can be realized through a one-step method, medicine stopping is not needed, a great number of clinical experiments prove that the detection result is accurate and reliable, and the method is very worthy of clinical large-scale popularization and application.
In the process of carrying out quantitative detection on renin activity and synchronous detection on the hypertension therapeutic drugs affecting ARR, the application uses a protein precipitation method to carry out pretreatment on a sample, and synchronously realizes effective extraction of angiotensin I and the hypertension therapeutic drugs while carrying out protein precipitation on the sample; and then, by utilizing the high-flux characteristic of the liquid chromatography tandem mass spectrometry technology, the extracted sample is synchronously detected by multiple indexes, and forty three therapeutic drugs affecting ARR are effectively screened while the content of angiotensin I is detected. ARR detection values are jointly analyzed in combination with interference drug screening results, so that the patient is prevented from stopping drug treatment, meanwhile, the false positive or false negative results of detection are effectively reduced, and the ARR judgment accuracy is improved.
The detection method provided by the application has the advantages that the required sample size is small, the angiotensin I content in the plasma before incubation can be detected by 100 mu L of plasma sample, and various antihypertensive drugs affecting ARR values can be qualitatively screened.
According to the pretreatment of the detection method provided by the application, the solution to be detected can be obtained only through protein precipitation, complex liquid-liquid extraction or SPE (solid phase extraction) process is not needed, the reduction of extraction rate of screening medicines with different characteristics caused by complex pretreatment is avoided, and the high-throughput treatment and detection of samples can be realized.
Further, the incubation means that after the sample is added into the generating buffer solution and mixed uniformly, the sample is put into a water bath at 37 ℃ for 3 hours; the non-incubated samples were used for detection of 43 hypertensive therapeutic agents affecting ARR; and the mobile phase A of the liquid phase is 0.1% formic acid aqueous solution, and the mobile phase B of the liquid phase is 0.1% formic acid methanol solution by volume ratio, and gradient elution is carried out.
Further, in the mass spectrometry detection, a triple quadrupole mass spectrometer is adopted, the model of the instrument is SCIEX 4500MD, and the mass spectrometry detection is carried out by adopting a positive ion mode (ESI+) of an electrospray ion source and a multi-reaction monitoring MRM mode.
The detection method provided by the application has the advantages that the liquid quality analysis time of each sample is only 3.8 minutes, and the detection efficiency is high.
And comparing the medicine which is positive according to the screening result of the mass spectrum MRM parameter with the retention time of the medicine standard in the screening medicine mixed working solution, and ensuring the accuracy of the positive screening result.
On the other hand, the application provides a detection method of the activity of renin, which mainly adopts qualitative screening of the antihypertensive drugs influencing ARR by detecting the generation rate of angiotensin I in a plasma sample and analyzing and judging the actual content of the activity of renin according to the detection result.
In still another aspect, the application provides a method for detecting the renin activity of a plasma sample by liquid chromatography tandem mass spectrometry and synchronously detecting 43 hypertensive drugs affecting ARR, for clinically judging the ARR value and the actual renin activity content.
Any one of the beta-blocker, the central alpha 2 receptor agonist and the nonsteroidal anti-inflammatory drug can reduce the activity of the renin of the patient; potassium-excreting diuretics or potassium-retaining diuretics can increase the renin activity of the patient; any one of an angiotensin converting enzyme inhibitor, an angiotensin receptor antagonist and a calcium ion antagonist can increase the renin activity of the patient. Therefore, the method for detecting the renin activity of the plasma sample and synchronously detecting 43 hypertensive drugs affecting the ARR through the liquid chromatography-tandem mass spectrometry can provide more accurate judgment for the actual renin activity besides qualitatively judging the ARR value under the condition of avoiding stopping the drug.
The application has the following beneficial effects:
(1) The qualitative screening of 43 hypertensive therapeutic drugs affecting ARR values is realized while the detection of renin activity (PRA) is carried out, and the ARR detection values are jointly analyzed by interfering with the drug screening results, so that the false positive or false negative results of the ARR detection are effectively reduced while the stopping of drug treatment of patients is avoided, the accuracy of the actual clinical renin activity detection is improved, and the clinical popularization and application are facilitated.
(2) In the process of detecting the activity of renin, a protein precipitation method is utilized to pretreat a sample, and the effective extraction of angiotensin I and 43 antihypertensive drugs can be synchronously realized while the protein precipitation is carried out on the sample, so that synchronous detection is carried out. The solution to be detected can be obtained only through protein precipitation in the pretreatment, and the complex liquid-liquid extraction or SPE process is not needed, so that the reduction of the extraction rate of screening medicines with different characteristics caused by the complex pretreatment is avoided.
(3) By utilizing the characteristics of high flux, high specificity and high sensitivity of the liquid chromatography tandem mass spectrometry technology, the extracted sample is synchronously detected by multiple indexes, and forty three therapeutic drugs affecting ARR are effectively screened while the content of angiotensin I is detected.
(4) The required sample size is small, and 100 mu L of plasma sample can detect the angiotensin I content in the plasma before incubation and qualitatively screen 43 antihypertensive drugs affecting ARR values.
(5) In the detection process, the MRM mass spectrum parameters of the screened medicines are utilized to perform primary screening of the therapeutic medicines while the quantitative detection of the angiotensin I is performed, and the retention time of each therapeutic medicine in the screening medicine mixed working solution is controlled to perform rechecking of the positive detection result so as to ensure the accuracy of the medicine screening result.
(6) The liquid quality analysis time of each sample is only 3.8 minutes, and the detection efficiency is high.
(7) Can provide a high-efficiency and accurate screening mode for screening the protoaldosis therapeutic drugs and a practical and reliable basis for clinical diagnosis.
Drawings
FIG. 1 is a sample detection flow chart in embodiment 1
FIGS. 2-13 are detection patterns of 43 kinds of ARR-affecting hypertensive drugs in example 1
FIG. 14 is a graph showing the detection of aldosterone in example 1
FIG. 15 is a plasma orthoaldehyde screening report of example 1
Detailed Description
The application will be described in further detail below with reference to the drawings and examples, it being noted that the examples described below are intended to facilitate an understanding of the application and are not intended to limit the application in any way. The reagents used in this example are all known products and are obtained by purchasing commercially available products.
Example 1: the application provides a qualitative detection method of ARR
1. Preparing a solution:
ZnSO 4 preparing a solution: weigh 3.56 grams ZnSO 4 .7H 2 O, dissolving in 40mL of water to prepare 89mg/mL ZnSO 4 .7H 2 O aqueous solution. The volume can be adjusted according to the requirement, and the product can be stored at room temperature.
Generating buffer solution: 12.11g of TRIS (hydroxymethyl) aminomethane (TRIS) and 7.4g of EDTA were placed in a 100mL volumetric flask, deionized water was added to 90mL, and the flask was sonicated for 30min until dissolution was uniform. Adding deionized water to the scale mark, and mixing. Transfer to a polypropylene storage container. Regulating pH to 5.45-5.50 with acetic acid, and storing at-20deg.C. On the day of assay, 100. Mu.L of 100mM PMSF (0.174 g PMSF in 10mL methanol) was added to 10mL of the production buffer to prepare a production buffer (pH 5.4-5.6).
Reaction termination liquid: formic acid or acetic acid.
Protein precipitants containing internal standard: 50 mu L of 1 mu g/mL of angiotensin I internal standard stock solution (methanol: water 1:1) and 100 mu L of 89mg/mL of zinc sulfate (ZnSO4.7H2O) aqueous solution are sucked, placed in a 10mL centrifuge tube, 9850 mu L of methanol is added into the centrifuge tube, and the mixture is uniformly mixed, so that the protein precipitant containing the angiotensin I internal standard is obtained.
Screening a drug mixed working solution: 43 drug standard substances to be tested and part of metabolite standard substances are dissolved in DMSO to prepare corresponding mother solutions, and the mixed working solution of the screened drugs with the limited concentration is prepared according to the limited concentration in the drug screening list and the limited concentration list affecting ARR values.
Second, sample detection:
the detection flow chart is shown in fig. 1.
(1) Reagent preparation: first, 20. Mu.L of the production buffer was added to two clean 1.2mL 96-well collection plates for subsequent sample pretreatment.
(2) Thawing a sample: the plasma sample to be tested is thawed in ice water (0 ℃) until it is thawed.
(3) Sampling: 2 100 μl aliquots of plasma samples were transferred in parallel to the two plates prepared in step (1) and the non-incubated and incubated samples were tested, respectively, and the remaining samples were immediately frozen at-20deg.C, wherein drug screening was performed using the non-incubated samples.
(4) Treatment of the incubated samples: sealing one batch of samples in the step (3) by using a silica gel pad, carrying out short vortex, then placing the samples in a water bath at 37 ℃ for 3 hours, adding 12 mu L of reaction stopping solution formic acid after 3 hours, carrying out vortex, and then adding 200 mu L of internal standard ANG I containing 5ng/mL of angiotensin I isotope 13 C 15 N internal standard working solution, vortex mixing, centrifuging at 15000rpm for 10 min at 4deg.C, collecting supernatant 100 μl, adding into 96-well sample plate, and performing liquid chromatography tandem mass spectrometry analysis (extensive study data prove that the drug screening process can not use incubation sample, detection result is inaccurate or difficult to detect, specific experimental data is omitted, and the incubation sample is only used for calculation of renin activity).
(5) Sample treatment without incubation: sealing the other batch of samples left in the step (3) by using a sealing gasket, and then uniformly vortex and mix. Immediately adding 12 mu L of reaction stopping solution formic acid, swirling, adding 200 mu L of internal standard working solution containing an angiotensin I isotope internal standard, swirling and uniformly mixing, centrifuging at 15000rpm at 4 ℃ for 10 minutes, taking 100 mu L of supernatant, adding 96-well sample plates, and carrying out sample injection analysis by a liquid chromatography tandem mass spectrometer (the drug screening process adopts an un-incubated sample of the step, and the un-incubated sample is used for calculating the activity of renin and can also be used for screening drugs interfering ARR).
(6) Calculation of renin Activity: renin activity is equal to the rate of production of angiotensin I in ng/mL/hr, calculated as: (angiotensin I concentration after incubation-angiotensin I concentration before incubation)/incubation time.
(7) When the liquid chromatography tandem mass spectrometry analysis is carried out, the liquid chromatography adopts gradient elution, and the reverse phase chromatography establishes the separation conditions of the to-be-detected object as follows: the column was Phenomnex C18 (2.6 μm, 50X 2.1 mm), the flow rate was 0.6mL/min, the column temperature was 40 ℃; wherein the mobile phase A is formic acid aqueous solution with the volume ratio of 0.1%, the mobile phase B is methanol solution with the volume ratio of 0.1%, and the volume ratio of the mobile phase A to the mobile phase B is 90-5%: 10-95%. Gradient procedure as shown in table 1, the retention times of angiotensin I and its isotopic internal standard are respectively: 1.92min.
TABLE 1 gradient elution procedure
Time (min) Flow rate (mL/min) Mobile phase a (%) Mobile phase B (%)
0 0.6 90 10
1 0.6 90 10
2 0.6 5 95
3.2 0.6 5 95
3.25 0.6 90 10
3.8 0.6 90 10
The retention time of the standard substance of each hypertension treatment drug in the "screening drug mixed working solution" is shown in table 2:
table 2 retention time of standard of antihypertensive drugs
When mass spectrometry detection is carried out, a triple quadrupole mass spectrometer is adopted for carrying out angiotensin I quantitative detection and depressurization drug qualitative screening before and after incubation, the model of the instrument is SCIEX 4500MD, an electrospray ion source positive ion mode (ESI+) and a multi-reaction monitoring MRM mode are adopted for carrying out mass spectrometry detection, and the corresponding mass spectrometry detection method is set as shown in tables 3 and 4:
table 3, mass spectrometry detection parameter settings
Table 4 mass spectrum parameters of each test object
The detection patterns of 43 hypertensive drugs affecting ARR are shown in figures 2-13, wherein figure 2 is the detection pattern of potassium-retaining diuretics Amiloride (Amiloride), CCB drugs Amiloride (amilopine), beta receptor blocker alolol (Arotinolol) and non-steroidal anti-inflammatory drugs Aspirin (Aspirin); FIG. 3 is a graph showing the detection of the beta blocker Atenolol, the ACEI drug Benazepril (Benazepril), the CCB drug Benidipine (Benidipine) and the beta blocker Bei Suoluo mol (Bisoprolol); FIG. 4 is a graph showing the detection of the potassium-excreting diuretics Bumetanide (Bumetanide), the ARB drug Candesartan (Candesartan), the ACEI drug Captopril (Captopril) and the ACEI drug Enalapril (Enalapril); FIG. 5 is a graph showing the detection of the potassium-retaining diuretics Eplerenone (Eplerenone), beta blocker Esmolol (Esmolol), the CCB drug Felodipine (Felodipine) and the CCB drug Flunarizine (Flunarizine); FIG. 6 is a graph showing the detection of the ACEI drug Fosinopril (Fosinopril), the potassium-excreting diuretic Furosemide (Furosemide), the potassium-excreting diuretic Hydrochlorothiazide (Hydrocholothiazide) and the ACEI drug Imidapril (Imidapiril); FIG. 7 is a graph showing the detection of the potassium-excreting diuretics Indapamide (Indapamide), ARB drug Irbesartan (Irbesartan), beta blocker Labetolol (Labetolol) and CCB drug Lercanidipine (Lercandipine); FIG. 8 is a graph showing the detection of ACEI drug Lisinopril (Lisinopril), ARB drug Losartan (Losartan), central alpha 2 receptor agonist Methyldopa (Methylldopa) and beta receptor blocker Metoprolol (Metoprol); FIG. 9 is a graph showing the detection of the beta blocker Nebivolol (Nebivolol), the CCB drug Nifedipine metabolite (Nifedipine), the CCB drug Nifedipine (Nifedipine) and the CCB drug Nicardipine (Nicardipine); FIG. 10 is a graph showing the detection of the ARB drugs Olmesartan (Olmesartan), the CCB drug Nimodipine (Nimodipine), the beta blocker Propranolol (Propranolol) and the ACEI drug Perindopril (Perindopril); FIG. 11 is a graph showing the detection of the ACEI drugs Ramipril (Ramipril), ACEI drug Quinapril (Quinapril), the beta receptor blocker Sotalol (Sotalol) and the potassium-retaining diuretic Spironolactone (Spironolide); FIG. 12 is a graph showing the detection of the potassium-retaining diuretic spirolactone (Spironolactone) metabolite, the ARB drug Telmisartan (telmiartan), the potassium-expelling diuretic Torasemide (Torasemide) and the ACEI drug Trandolapril (Trandolparil); FIG. 13 shows the detection patterns of the ARB drugs Valsartan (Valsartan), angiotensin I (Ang I) and the angiotensin I isotope internal standard (Ang I13C 15N). The X, Y axis coordinates of the detection patterns of 43 kinds of hypertension drugs affecting ARR are consistent with the X, Y axis coordinates of the detection pattern of the potassium-retaining diuretic Amiloride (Amiloride) in fig. 2.
(8) Detection of aldosterone: the sample pretreatment process for detecting aldosterone in plasma adopts a carrier liquid-liquid extraction method for extraction, adopts a liquid chromatography tandem mass spectrometry method for detection, and comprises the following specific steps: transfer 300-450uL plasma, add 50uL aldosterone internal standard solution, load onto SLE plate, stand for ten minutes, and elute with ethyl acetate and n-hexane mixed solvent (1:1) 1.5 mL. After the eluent is dried, re-dissolving the sample, detecting by using a liquid chromatography tandem mass spectrometer, and adopting an ESI+ detection mode, wherein the quantitative MRM ion pair of the Aldosterone Aldriterone is 361.3/315.1; the MRM ion pair of the Aldosterone internal standard Aldriterone-d 8 is 369.4/323.1; a representative detection spectrum is shown in fig. 14. A large number of researches prove that the pretreatment and detection processes of the aldosterone cannot synchronously screen 43 drugs affecting ARR, the detection result is inaccurate or difficult to detect, and the reason is probably that the pretreatment process is not suitable for extracting 43 drugs affecting ARR, and experimental data are slightly available.
(9) Drug screening detection report: in the plasma orthoaldehyde screening report (shown in fig. 15), the results of screening the drug factors which cause false positive or false negative of ARR are summarized, and the results of screening the ARR and the drug are combined clinically, so that the false positive or false negative results caused by the drug interference can be effectively eliminated.
Example 2: false negative clinical sample detection verification
In this example, the detection and qualitative determination of ARR were performed on clinical samples using the detection method as provided in example 1, and the detection results of the clinical samples are shown in table 5.
TABLE 5 clinical sample test results
Based on the above detection results of the sample, ARR is less than the tangent point value (30) for disease diagnosis, and therefore, the patient of this sample should be judged negative. However, in combination with the screening results of the patient antihypertensive drugs in Table 6, ARB and CCB drugs tested positive, both of which resulted in false negatives of the ARR test, a comprehensive evaluation, and even though the ARR results of this patient sample were below the cut-point value, it was still possible to be a positive patient, suggesting further diagnostic experiments.
Table 6, patient drug screening results
Drug factors Screening results Drug name Effects on aldosterone Effects on renin Effects on ARR
Beta-blockers - Without any means for ↓ ↓↑ ∈ (false positive)
Central alpha 2 receptor blockers - Without any means for ↓ ↓↓ ∈ (false positive)
Non-steroidal anti-inflammatory drugs - Without any means for ↓ ↓↓ ∈ (false positive)
Potassium-expelling diuretic - Without any means for →↑ ↑↑ ∈ (false negative)
Potassium retention diuretic - Without any means for ↑ ↑↑ ∈ (false negative)
ACEI - Without any means for ↓ ↑↑ ∈ (false negative)
ARBs + Valsartan (Valsartan) ↓ ↑↑ ∈ (false negative)
Dihydropyridines CCBs + Amlodipine →↓ ↑ ∈ (false negative)
The patient is indeed a positive patient for primary aldosteronism, which is confirmed by a captopril diagnosis test.
Example 3: false positive clinical sample detection verification
In this example, the detection and qualitative determination of ARR were performed on clinical samples using the detection method as provided in example 1, and the detection results of the clinical samples are shown in table 7.
TABLE 7 clinical sample test results
Based on the above detection results of the sample, ARR is greater than the tangent point value (30) for disease diagnosis, and thus the patient of this sample should be judged positive. However, in combination with the screening results of the patient antihypertensive drugs in Table 8, the beta-blocker and the central alpha 2 receptor agonist were tested positive, both of which resulted in false positives for ARR testing, a comprehensive evaluation, although the ARR results for this patient sample were above the cut-point value, and further diagnostic experiments were suggested.
Table 8, patient drug screening results
Drug factors Screening results Drug name Effects on aldosterone Effects on renin Effects on ARR
Beta-blockers + Metoprolol ↓ ↓↓ ∈ (false positive)
Central alpha 2 receptor blockers + Methyldopa ↓ ↓↓ ∈ (false positive)
Non-steroidal anti-inflammatory drugs - - ↓ ↓↓ ∈ (false positive)
Potassium-expelling diuretic - - →↑ ↑↑ ∈ (false negative)
Potassium retention diuretic - - ↑ ↑↑ ∈ (false negative)
ACEI - - ↓ ↑↑ ∈ (false negative)
ARBs - - ↓ ↑↑ ∈ (false negative)
Dihydropyridines CCBs - - →↓ ↑ ∈ (false negative)
The positive diagnosis experiment of captopril proves that the patient is a negative patient.
Example 4: positive clinical sample detection and validation
In this example, the detection and qualitative determination of ARR were performed on clinical samples using the detection method as provided in example 1, and the detection results of the clinical samples are shown in table 9.
TABLE 9 clinical sample test results
Based on the above detection results of the sample, ARR is greater than the tangent point value (30) for disease diagnosis, and thus the patient of this sample should be judged positive. Meanwhile, the screening results of the antihypertensive drugs of the patients in the table 10 are combined, the drugs of the ARB and the potassium-excreting diuretic drugs are detected positive, the ARB and the potassium-excreting diuretic drugs can both cause false negatives of the ARR detection, the comprehensive evaluation is carried out, after the patient sample takes the ARB and the potassium-excreting diuretic drugs which can cause the false negatives, the ARR result is still higher than the tangent point value, the patient can be a positive sample, and the diagnosis experiment can be carried out without stopping the drugs.
Table 10 results of drug screening for patients
Drug factors Screening results Drug name Effects on aldosterone Effects on renin Effects on ARR
Beta-blockers - - ↓ ↓↓ ∈ (false positive)
Central alpha 2 receptor blockers - - ↓ ↓↓ ∈ (false positive)
Non-steroidal anti-inflammatory drugs - - ↓ ↓↓ ∈ (false positive)
Potassium-expelling diuretic + Hydrochlorothiazide →↑ ↑↑ ∈ (false negative)
Potassium retention diuretic - - ↑ ↑↑ ∈ (false negative)
ACEI - - ↓ ↑↑ ∈ (false negative)
ARBs + Candesartan cilexetil ↓ ↑↑ ∈ (false negative)
Dihydropyridines CCBs - - →↓ ↑ ∈ (false negative)
Although the present application is disclosed above, the present application is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the application, and the scope of the application should be assessed accordingly to that of the appended claims.

Claims (6)

1. The synchronous qualitative detection method for the antihypertensive drugs affecting ARR in the process of detecting the activity of renin by a liquid chromatography-tandem mass spectrometry method is characterized in that the method can be used for judging and analyzing the value of ARR by combining the concentration of aldosterone by detecting the generation rate of angiotensin I in a plasma sample and the concentration of the antihypertensive drugs affecting ARR, and analyzing and judging the ARR to be one of negative, positive, false negative or false positive; the total of 43 hypertension treatment medicines affecting ARR are respectively:
nine beta-blockers include alolol, atenolol, bisoprolol, esmolol, labetalol, metoprolol, nebivolol, propranolol, sotalol;
five kinds of potassium-expelling diuretics include bumetanide, furosemide, hydrochlorothiazide, indapamide and torsemide;
the three potassium-retaining diuretics include amiloride, eplerenone and spironolactone;
ten angiotensin converting enzyme inhibitors include benazepril, captopril, enalapril, fosinopril, imidapril, lisinopril, perindopril, quinapril, ramipril, trandolapril;
six angiotensin receptor antagonists include candesartan, irbesartan, losartan, olmesartan, telmisartan, valsartan;
eight calcium antagonists include amlodipine, benidipine, felodipine, flunarizine, lercanidipine, nicardipine, nifedipine, nimodipine;
a central α2 receptor agonist comprises methyldopa;
a non-steroidal anti-inflammatory drug comprising aspirin; the ARR has a calculation formula as follows: ARR = aldosterone concentration/angiotensin i production rate;
the beta-receptor blocker, the central alpha 2 receptor agonist and the nonsteroidal anti-inflammatory drug can all cause ARR to rise and cause false positive results to appear; the potassium-expelling diuretic, potassium-preserving diuretic, angiotensin converting enzyme inhibitor, angiotensin receptor antagonist and calcium ion antagonist can reduce ARR, and false negative result appears;
the step of analyzing the value of ARR is: analyzing and judging ARR as one of negative, positive, false negative or false positive through a clinical medication AI guiding system; the clinical medication AI guiding system comprises a patient information module, a database module and a decision tree system;
the analysis and judgment method of the decision tree system comprises the following steps: when the detection result of the ARR is less than 30, if the patient contains the drug capable of reducing the ARR, the patient can be judged to be false negative, and further diagnosis experiments or drug withdrawal checks are required; when the ARR detection result is less than 30, if the patient contains the medicine capable of raising the ARR, the patient can be judged as negative; when the detection result of the ARR is more than 30, if the medicine capable of increasing the ARR is detected in the patient at the same time, the patient can be judged to be false positive, and further diagnosis experiment or medicine stopping check is needed; when the ARR detection result is more than 30, if the patient contains the drug capable of reducing the ARR, the patient can be judged to be positive;
the non-incubated samples were used for detection of 43 hypertensive therapeutic agents affecting ARR; the mobile phase A of the liquid phase is 0.1% formic acid aqueous solution, the mobile phase B of the liquid phase is 0.1% formic acid methanol solution by volume ratio, the gradient procedure is shown in the following table,
the detection of the aldosterone adopts a carrier liquid-liquid extraction method for extraction, and adopts a liquid chromatography-tandem mass spectrometry method for detection.
2. The method of claim 1, wherein the information module is configured to record basic information of the patient; the database module is used for storing the detection result of ARR and the detection result of the hypertension treatment drug; and the decision tree system automatically analyzes and judges the ARR index as one of negative, positive, false negative or false positive according to the detection result.
3. The method of claim 2, wherein the rate of angiotensin i production is determined by measuring the concentration of angiotensin i in the pre-incubation sample and the post-incubation sample according to the formula: angiotensin i production rate = (angiotensin i concentration after incubation-angiotensin i concentration before incubation)/incubation time.
4. The method according to claim 3, wherein the specific detecting steps are as follows:
(1) Sample pretreatment: 1) Taking two 100mL samples to be tested in parallel, respectively adding a generating buffer solution into the samples, uniformly mixing, incubating one sample, and directly carrying out the next operation without incubating the other sample; 2) Adding a reaction stopping solution into the sample added with the generated buffer solution and uniformly mixing; 3) Adding an internal standard-containing protein precipitant into the sample added with the reaction stopping solution in the step 2) and uniformly mixing by vortex; 4) Uniformly mixing the sample added with the protein precipitant in the step 3), centrifuging, and taking supernatant for machine detection;
(2) Detecting angiotensin I and screening 43 antihypertensive drugs affecting ARR by adopting a high performance liquid chromatography tandem mass spectrometry system to the solution to be detected of the sample before incubation;
(3) For the sample with the positive drug according to the mass spectrum parameter screening result in the step (2), the retention time of the standard substance of each hypertension therapeutic drug in the screening drug mixed working solution is required to be simultaneously compared, whether the drug screening result is false positive or not is checked, and the accuracy of the drug screening positive result is ensured from two dimensions of the mass spectrum parameter and the retention time;
the protein precipitant containing the internal standard is as follows: methanol, zinc sulfate aqueous solution and an angiotensin I isotope internal standard are prepared into a protein sedimentation agent containing the internal standard;
the generating buffer solution is as follows: tris and EDTA buffers containing the angiotensin converting enzyme inhibitor PMSF (phenylmethylsulfonyl fluoride);
the reaction termination liquid is as follows: formic acid or acetic acid;
the screening medicine mixed working solution comprises the following components: the mixed working solution of the screening medicaments for detecting the lower limit concentration is prepared by using 43 standard substances of the hypertension therapeutic medicaments affecting the ARR.
5. The method according to claim 4, wherein the incubation is performed by adding the sample to the buffer and mixing the sample with the buffer, and then placing the sample in a water bath at 37 ℃ for 3 hours;
the retention time of the standard substance of each hypertension treatment drug in the screening drug mixed working solution is shown in the following table:
。
6. the method of claim 5, wherein the mass spectrometry is performed using a triple quadrupole mass spectrometer having a model SCIEX 4500MD, using positive ion mode (esi+) of electrospray ion source and multi-reaction monitoring MRM mode, and the corresponding mass spectrometry is set forth in the following table:
the mass spectrum parameters of each test object are shown in the following table:
。
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Publication number Priority date Publication date Assignee Title
CN112014509A (en) * 2020-09-03 2020-12-01 复旦大学附属中山医院 Method for synchronously determining angiotensin I and aldosterone in sample
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112014509A (en) * 2020-09-03 2020-12-01 复旦大学附属中山医院 Method for synchronously determining angiotensin I and aldosterone in sample
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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
基于血浆醛固酮/肾素浓度比的联合策略在原发性醛固酮增多症中的筛查价值;孙明芳 等;《军事医学》;20150430;第39卷(第4期);279-283 *

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